Principles of Proteomics overview Lim Wee Guan, PhD. Product Specialist

Principles of Proteomics
overview
Lim Wee Guan, PhD.
Product Specialist
Life Science Group
Southeast Asia and Taiwan
One Genome- Different Proteomes
Life is base on proteins and their
interactions
Why proteome research?
Genome
Gene
Transcriptional control
RNA
Degradation
mRNA
Degradation
Pre-Protein
Degradation
Protein
Degradation
Translational control
Posttranslational
Modifications
• Low correlation between mRNA abundance and protein level
• Gene sequence does not code for post-translational modifications
• Genome is static; Proteome reflects the dynamic nature of cellular
processes
What is proteomics?
Proteomics is the study of protein
characteristics and functions to obtain a
global integrated view of:
• normal and abnormal cellular processes
• protein-protein interactions
• regulatory networks
The definition of all proteins of interest within a biological
sample.
Why study proteomics?
Mining: identification of all proteins in a sample
Protein Expression Profiling: identification of protein in a
sample as a function of a state of the organism or cell
under certain condition
Protein Network Mapping: Approach to determine how
proteins interact with each other in living systems.
Mapping of protein modifications: identification how and
where proteins are modified.
The Question
Cell specific expression
Culture conditions
Metabolic state
Drugs
Temperature
Dysfunctions, Cancer
Stress
Simultaneous protein profiling to understand the function
and regulation of thousands of proteins
PROTEOMICS
Structural
Proteomics
Expression Proteomics
Functional
Proteomics
• Compares patterns of proteins expressed in a
given tissue or cell under certain conditions
• Quantitative analysis and identification of
proteins
• Ultimately maps these changes back to the
underlying or corresponding gene
Key concern of proteomics
The Expression Proteomics Challenge is…
…the complexity of a cell and the diverse nature of proteins:
Number of diff proteins in cells/tissue/organism
Humans have ~ 30,000 to 40,000 genes
Estimated 100,000 -300,000 human protein
count
Dynamic range of proteins
Expression levels vary
Post-translational modifications
Chemical diversity of protein
-size, pI/charge, hydrophobicity,etc
Key concern of proteomics
How to reduce the complexity of samples
Separate the proteins in an electrical field: electrophoresis
Electrophoresis using 2 different properties
By isoelectric point (pI): IEF
IEF
By size (MW): SDS-PAGE
2-D Electrophoresis
Proteins
migrate
through the
gel at a rate
inversely
proportional
to their size.
Smallest
proteins travel
the furthest
distance
Isoelectric Focusing (IEF) with IPG strips
70 - 2 40 m m
3 -4 m m
+
ge l
pH 3
Pla s tic b a c k in g
–
pH 7.5
pH 10
–
+
pH 3
pH 7.5
–
+
pH 3
pH 7.5
pH 10
–
+
pH 3
pH 10
pH 7.5
pH 10
2D proteomics for maximum resolution
pH 3
pH 3
pH 7.5
+
pH 7.5
MW
–pH10
pI
–
pH10
size
charge
Combine IEF and SDS-PAGE to get maximum separation
Birth of 2D!!!
What is the protein expression pattern at a particular point in time?
Cell specific expression
Culture conditions
Metabolic state
Drugs
Temperature
Dysfunctions, Cancer
Stress
Simultaneous protein profiling to understand the function
and regulation of thousands of proteins
Advantage of 2D
• Global view of protein expression, ability to find signaling
cascade.
• Ability to detect PTM
• Direct correlation of protein expression to disease
• Quantitative analysis of protein regulation
• Routinely separate 3000 spots per gel
• Mass spect compatible
2-D Expression Proteomics Methodology
FIRST DIMENSION
3
SAMPLE PREPARATION
4
pH
7
10
-
+
-
+
Condition A
Condition B
MASS SPECTROMETRIC
IDENTIFICATION OF SPOTS
STAINING
ANALYSIS &
EXTRACTION
IMAGING
SECOND
DIMENSION
2-D Expression Proteomics Tools
Sample Preparation
1st dimension IEF
2nd dimension SDS-PAGE
MS
Gel staining
Spot excision
Imaging and Analysis
The next steps…
Destain and Tryptic Digest
• Cleave at known residues
• Optimal length for MS Analysis
6-20 amino acid sequences
• Widely used for database lookups
Mass Spec Workflow
What do scientists want from Mass Spec data?
Data on:
• peptide masses
• peptide fragmentation
What makes good data?
• sensitivity
• resolution
• mass accuracy
What does Mass Spec data look like?
What do scientists do with Mass Spec data?
• Peptide masses are compared with real database values
• Peptide Mass Fingerprinting allows proteins to be identified for
amino acid sequence
• Software is Key:
1. Controlling the systems
2. Probability and Statistical Algorithms
3. Organization of Data
Protein is Identified…Now What?
Experimental Timeframe
2D PAGE separation, Image analysis and
Protein excision
1. Rehydration of first dimension gels: 12 hours
2. Isoelectric focusing: 2 - 16 hours (depending
on gel length and pI range to be analyzed)
3. Equilibration and SDS-PAGE separation: 2 – 8
hours (depending on gel size)
4. Gel fixation and staining: 4 hours to overnight
5. Image analysis: 1 – 5 days / comparison set
(depending on gel number, protein number)
6. Protein excision: 3 hours – 1 day (depending
on protein number)
Total: 5 – 10 days
Mass Spectrometry:
1. Protein digestion: 7 hours
(overnight)
2. Sample concentration: 0.25 - 0.5
day
3. Sample Loading: 0.25 - 0.5 day
4. MALDI MS analysis; spectra
storage: 0.75 days to overnight
5. Spectra interpretation: 1 day
6. Database submission: 1 - 4
days (manual)
7. Protein identification: 5 - 7 days.
Total: 9.5- 15 days
Once the protein sample has been prepared, 2D PAGE separation, MS
analysis and subsequent protein identification usually takes between
15-25 days.
Research
Research
Sample prep
FIRST DIMENSION
pH
3
SAMPLE PREPARATION
4
7
10
-
+
-
+
Condition A
Condition B
MASS SPECTROMETRIC
IDENTIFICATION OF SPOTS
STAINING
ANALYSIS &
EXTRACTION
IMAGING
SECOND
DIMENSION
What is sample prep?
Sample preparation involves everything that lies between the sample and 1st
dimension IEF
Sample
Protein mixture
•
Cell disruption/ lysis
•
Protein extraction, solubilization
•
Contaminant removal/ cleanup
•
Fractionation
The Equation
• Good sample preparation
=
=
Good
2D Gels
Bad sample prep = bad 2D gels
Sample Preparation
Tools for Sample Cleanup and Fractionation
Sample Preparation
Sample prep requirements
• Reproducibly solubilize proteins of all classes,
including hydrophobic proteins
• Prevent protein aggregation and loss of solubility
during isoelectric focusing
• Yield proteins of interest at detectable levels, which
may require the removal of interfering abundant
proteins or non-relevant classes of protein
2 Sample Prep Strategies :
General Purpose Cleanup
• Improve Resolution
• Improve Reproducibility
Fractionation
• Reduce Complexity
• Improve Range of Detection
• Enrich low-abundance proteins
Applicable to most sample types, applications
•Contaminant removal
Salts
Detergents, phenolics, etc.
Abundant proteins
•Disulfide bond removal
(reduction/ alkylation)
•Total protein extraction
Contaminant removal
Introduction of contaminants from……
Cell/ tissue lysis method
Sample source
Detergents
Nucleic acids
Salts
Lipids, sugars
Buffers
Phenolics
Insoluble material
Ions, small molecules
Abundant proteins
Need for contaminant removal
Problem: salt contamination
Horizontal streaking caused by excess salt (lysis method)
Problem: detergent contamination
Horizontal streaking caused by excess SDS (lysis method)
Solution: ReadyPrep 2-D Cleanup Kit
Untreated
Treated
E. coli cell lysates spiked with 1% SDS
Improved resolution, less streaking on 2-D gels
Removes salts & detergents, phenolic compounds, lipids, ionic
contaminants
>95% recovery
Concentrates dilute samples
Solution: Bio-Spin columns for buffer exchange
Size Exclusion Chromatography
•
Large molecules (proteins) pass around beads
•
Small molecules (salt) retained in beads
Requires buffer exchange with IEF buffer
Bio-Spin 6 and Micro Bio-Spin 6
• Bio-Spin: 50-100 ml sample
• Micro Bio-Spin: 10-75 ml sample
Problem: Albumin and IgG in serum samples
Abundant proteins impair detection of other proteins
Solution: Aurum Serum Protein and Affi-Gel Blue kits
Affinity chromatography
•
Albumin and/or IgG bind to column
(removed)
•
Other proteins pass through column
•
Some proteins bind to column due to
non-specific protein interactions with
albumin and IgG
Aurum Serum Protein and Affi-Gel Blue kits
Bead
Problem: Disulfide bond removal
•
•
•
•
Disulfide bridges within and between proteins cause spurious
spot patterns
Prevention: Reducing agents
A big problem in the alkaline region
Reducing agents tend to be negatively charged
• DTT migrates off of gel during IEF
Solution: Reduction followed by alkylation
•Reduction with TBP
•Alkylation with IAA (prevents
reformation of disulfide bonds)
•Prior to IEF
ReadyPrep Reduction/Alkylation kit
Mouse liver extract run on pH 7-10, 11 cm strips.
Cup loaded.
The challenge: see low expressed proteins
•
Estimated ~100,000 – 300,000 human protein count
•
Spot counts per gel typically 500 - 2000 depending on gel size and pI range
•
Sample load limits detection of low- abundance proteins
Need for enrichment
Strategy 1: Increase loading
50 µg E.Coli
500 µg E.Coli
100 µg E.Coli
1000 µg E.Coli
250 µg E.Coli
Strategy 2: Increase gel size
pH 5
7 cm
pH 8
8%
60 µg E. coli 944 spots
11 cm
16%
100 µg E. coli 1,287 spots
150 µg E. coli 1,724 spots
17 cm
Strategy 3: Narrow-range IPGs
pH 3-10
pH 3- 6
pH 5- 8
pH 7-10
Strategy 4: Fractionation
Separation of a protein sample into
fractions according to
Cellular location
Solubility/ hydrophobicity
Size (mw)
Charge/ pI
Strategy 4: Fractionation
Fractionation enables analysis of components
Reduced sample complexity
Increased resolution (more dimensions)
Increased load of proteins of interest
Enrichment of low-abundance proteins
Fractionation
Application-dependent
•Cellular location
•Biophysical property
•Solubility/ hydrophobicity
•Size
•Charge/ pI
Fractionation by cellular location
ReadyPrep Protein Extraction kits
Cytoplasmic/Nuclear
Isolates nuclei, extracts proteins
Membrane I
Triton X-114, temp.-dep. Partitioning
Membrane II
Carbonate extraction
Signal
Triton X-100 differential solubility
Caveoli-associated proteins
Membrane protein isolation
Signal
Membrane I
Membrane II
Mouse liver isolated with different kits.
~450 ug extract loaded onto 17 cm pH 3-10 IPG strips.
Total Protein
Fractionation by protein solubility
ReadyPrep Sequential Extraction Kit
sample
ReadyPrep Protein Extraction Kit
(Soluble/Insoluble)
sample
Soluble
proteins
Solution 1
Solution 2
Solution 3
SB 3-10
ASB14
Insoluble
proteins
Benefits of these approaches
• Enrichment of proteins of interest
• Well-understood protocols, in kit format
Reproducibility
Ease-of-use
Easy to try a number of approaches
Fractionation by size: Model 491 prep cell
Effective visualization of
•Protein families
•Post translational modifications
Fractionation by size: Bio-Spin 30
Size Exclusion Chromatography
•Large molecules (proteins) pass around beads
•Small molecules retained in beads
Bio-Spin 6 and Micro Bio-Spin 30
• P30: >40 kDa
Additional chromatographic options
• Bio-Gels 2, 4, 10, 60, and 100
• Fractionation ranges from 2 -100 kDa
Fractionation by charge
Ion Exchange Chromatography
•
•
•
•
•
Positively charged proteins (basic) bind to CEX (cation exchange)
Negatively charged proteins (acidic) bind to AEX (anion exchange)
Elution with high salt, but pH gradient also effective
High dynamic binding capacity
Charge of protein is dependent on buffer conditions
Aurum AEX and CEX kits
pI=4
pI=9
-----
AEX
---
+
---
+
---
CEX
+
+
+
Fractionation by charge
Total
Protein
AEX
CEX
1
Acidic protein binds to AEX
not CEX
3
Basic protein binds to CEX
not AEX
5
Protein is enriched on CEX
4
Protein is enriched on AEX
Fractionation by pI
Removes proteins outside of pI range of interest
Pair with narrow- and micro-range IPGs (2DGE)
All performed in liquid medium
Particularly helpful with
• High molecular weight proteins
• Hydrophobic/ membrane proteins
• Extreme pIs
The Rotofor® cell
Fractionation by pI
MW
Direct 2-DE:
pH 3 - 6
pH 4 - 7
pH 5 - 8
70
50
35
30
15
8
Prefractionated 2-DE:
pH 3 - 6
pH 4 - 7
pH 5 - 8
70
50
35
30
15
CSF sample pH 1.5 - 4.5
CSF sample pH 4.5-6.0
CSF sample pH 6.0 –7.5
8
Sample preparation products
Sample preparation in proteomics
•
Critically important consideration in expression proteomics research
•
No single approach fits all
Sample type
Size, source
Research goal
Laboratory resources
Unique position
Bio-Rad has
an extensive product offering
for 2-D sample preparation products!
First Dimension Tools
ReadyPrep 2D starter kit:
•Rehydration buffer
•Equilibration buffer I
•Equilibration buffer II
•Glycerol solution
•Agraose overlay
•IAA
Sample prep tools
2-D Electrophoresis
Tools for Rapid, High-Resolution Protein Separations
2-D Electrophoresis
2-D Electrophoresis Workflow
Precast Gels
2-D Tools
Stains
Stainers
• Simplified staining ensures consistent results
• Improved gel handling
• Available in two sizes (small and large)
• Stain compatibility
Imaging and Analysis
Tools for Acquisition and Analysis of Protein Expression Data
PDQuest Software
Imaging instruments
Spot cutter instrument
Imaging
Detection capabilities
GS-800
Molecular Imager FX
Pro (Plus) Systems
VersDoc Model 4000
GS-800 Densitometer
VersaDoc Model
4000
Molecular Imager FX
Pro-Q Diamond
●
Pro-Q Emerald
●
Cy5
●
y2
●
Cy3
●
SYPRO Orange
●
●
SYPRO Red
●
●
SYPRO Ruby
●
●
Coomassie Blue
●
●
●
Copper
●
●
●
Silver
●
●
●
Zinc
●
●
●
Research
Assessment of acetaminophen toxicity in mouse liver
EXQuest Spot Cutter
• 2-D and 1-D spot cutting
•Gel matching and excision driven by PDQuest software
• High speed precision robotics
-Cuts 600 spots per hour
-Accuracy of +0.1 mm ensures accurate excision of
small, closely grouped spots
•Compatible with visible light and UV-based applications,
including SYPRO Ruby and Orange, silver, and
Coomassie Blue
• Cuts free or backed gels and PVDF membranes
2-D Expression Proteomics Tools
Sample Preparation
1st dimension IEF
2nd dimension SDS-PAGE
MS
Gel staining
Spot excision
Imaging and Analysis
Support
www.expressionproteomics.com
Integration with PDQuest
•PDQuest controls the imaging instruments
•PDQuest optimizes/normalizes images for
accurate comparison
•Analytical tools identify protein spots of
interest
•Advanced algorithms identify & match
protein patterns
•Controls the ProteomeWorks Plus spot cutter
•Stores, manages and records sample info
Discover with Bio-Rad!
•
Sample Preparation
Large and small scale
Electrophoretic, chromatographic, or chemical reagent based
Most complete line of sample prep devices and kits for proteomics available
•
Electrophoresis
7, 11, 17, 18, and 24 cm IPG strips for first dimension
Broad, non linear, narrow and micro pH range
Mini, midi, and large format precast gels and gel rigs for second dimension
Complete line of power supplies and reagents
•
Image Analysis
Densitometer, laser based, and a complete line of camera based imagers for every need
•
PDQuest 2-D Analysis Software
Functionality for all 2-D applications
Over 25 years of PDQuest 2-D Analysis Software assisted discoveries
300+ publications since 1976
Over 100 from 2002-present
•
Spot cutting
Fastest and most accurate instrument available
Integrated with full suite of expression proteomics products
Thank you